A major breakthrough in the study of the disease cystic fibrosis (CF) occurred two years ago when the gene defective in CF was identified and the nucleotide sequence of the corresponding CDNA molecular was determined. The protein encoded by this CDNA was termed the cystic fibrosis transmembrane conductance regulator (CFTR). Based upon the deduced amino acid sequence, CFTR is predicted to consist of two similar motifs, each containing six potential membrane spanning segments followed by a domain containing the features of known nucleotide-binding folds (NBFs). These repeated structural motifs are separated by a single hydrophilic domain having a highly charged nature. Comparison of the amino acid sequence of CFTR to protein databases indicated that it is related to the ATP-binding cassette (ABC) superfamily of transporter proteins found in a variety of prokaryotic and eukaryotic organisms. Most members of this family of proteins transport specific molecules across the plasma membrane, and retain significant amino acid homology over an extended region (roughly 200 amino acids) of their NBFs. Because of its homology to these proteins, CFTR may carry out a transport function in addition to its function as a chloride channel. Another member of this transporter superfamily is the yeast Ste6 protein, which exports the lipopeptide hormone a-factor from MATa yeast cells. The Ste6 protein retains significant sequence and structural homology to CFTR. By aligning the sequences of these and several other members of the transporter superfamily, we have identified specific amino acid residues within the Ste6 protein that correspond to residues mutated in individuals with CF. In preliminary experiments, we found that the analysis of naturally occurring CFTR mutations in the context of the Ste6 protein allows us to characterize the functional consequences of CFTR mutations on NBF (And possibly transporter) function. We propose to both expand this study and complement it with a domain exchange approach to further characterize the structure and function of the regions conserved between these proteins. We also intend to utilize the power of yeast molecular genetics to characterize the structure and function of CFTR directly. To do this, we constructed an expression system that allows the efficient synthesis of CFTR in yeast. We will use the yeast CFTR expression system to carry out a detailed molecular analysis of the structure, function, and topology of CFTR. In addition, we intend to set up a vesicle-based in vitro system to further explore the function of CFTR. This combination of approaches will allow us to directly characterize postulated functions of CFTR, both as a chloride channel and as a transporter. We will then determine how mutations in CFTR affect these functions. Together, these studies will increase our knowledge of CFTR function, allowing us to better understand how mutations in CFTR result in CF.